Epitope peptides derived from vascular endothelial growth factor receptor 1 and vaccines containing these peptides

ABSTRACT

The present invention provides immunogenic peptides comprising the amino acid sequence of SEQ ID NO: 1, 2, 13, 32, and peptides comprising the above-mentioned amino acid sequences in which 1, 2, or several amino acids are substituted or added, and having cytotoxic T cell inducibility, and also provides drugs for treating or preventing tumors comprising these peptides. The peptides of this invention can be used as vaccines.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. National Stage Application ofPCT/JP2006/303352, filed Feb. 17, 2006, which claims the benefit of U.S.Provisional Application Ser. No. 60/657,527 filed Feb. 28, 2005, all ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to peptides that are extremely effectiveas cancer vaccines, and drugs for treating and preventing tumors, whichcontain these peptides.

BACKGROUND OF THE INVENTION

Tumor growth is generally limited to 1˜2 mm³ in the absence of avascularized blood supply, and angiogenesis has a critical role in theinvasion, growth and metastasis of tumors (Folkman, J. (2002) Semin.Oncol. 29: 15-8, Folkman, J. (1996) Nat. Med. 2: 167-8, Kerbel andFolkma, (2002). Nature Rev. Cancer. 2: 727-39, Brown et al., (1995) Hum.Pathol. 26: 86-91, Eberhard et al., (2000) Cancer Res. 60: 1388-93.). Ithas been also shown that inhibition of tumor angiogenesis is associatedwith suppression of tumor progression. In order to achieve suppressionof angiogenesis, a number of investigators have been examiningtherapeutic strategies targeting vascular endothelial growth factor(VEGF) and VEGF receptor (VEGFR), which play critical roles inregulating the process of angiogenesis. These studies have shown thattumor growth can be successfully suppressed in vitro and in vivo usingmonoclonal antibodies, recombinant receptors or inhibitors for signaltransduction (El-Mousawi et al., (2003) J. Biol. Chem. 278: 46681-91,Stefanik et al., (2001) J. Neurooncol. 55: 91-100, Wood et al., (2000)Cancer Res. 60: 2178-89, Luttun et al., (2002) Nat. Med. 8: 831-40,Lyden et al., (2001) Nat. Med. 7: 1194-201, Lu et al., (2001) CancerRes. 61: 7002-8.). However, these strategies require frequent orcontinuous administration of the reagents at relatively high doselevels, which may be associated with significant inconvenience andadverse effects.

VEGF binds two related tyrosine kinase receptors, VEGFR1 (Flt-1) andVEGFR2 (KDR), which are strongly expressed on endothelial cells in tumortissue but not in normal tissue (Risau, W. (1997) Nature. 386: 671-4,Ferrara and Davis-Smyth, (1997) Endor. Rev. 18: 4-25, Shibuya et al.(1999) Curr. Topics. Microbiol. Immunol. 237: 59-83, Plate et al.,(1994) Int. J. Cancer. 59: 520-9.). VEGFR1 is the first VEGF receptor tobe identified (Shibuya et al., (1990) Oncogene 5: 519-24.), and itinteracts with VEGF (VEGF-A) and with two other members of VEGF family,VEGF-B (Olofsson et al., (1996) Proc. Natl. Acad. Sci. USA 93: 2576-81.)and placenta growth factor (P1GF) (Maglione et al., 1991. Proc. Natl.Acad. Sci. USA 88: 9267-71.). By displacing VEGF from VEGFR1, P1GF isexpected to make more VEGF available to bind and activate VEGFR2 andthereby enhance VEGF-driven angiogenesis (Park et al., (1994) J. Biol.Chem. 269: 25646-54.). Other studies have shown that a synergism existsbetween VEGF and P1GF in vivo, especially during pathologicalsituations, as evidenced by impaired tumorigenesis and vascular leakagein P1GF−/− mice (Carmeliet et al., (2001) Nat. Med. 7: 575-83.).

Recent reports have shown that vaccination using cDNA or recombinantprotein of mouse VEGFR2 is associated with significant anti-tumoreffects in mouse tumor models (Li et al., (2002) J. Exp. Med. 195:1575-84, Niethammer et al., (2002) Nat. Med. 8: 1369-75.). But theseresults cannot directly warrant clinical application of this strategy,since they used the mouse homologue of human VEGFR2 in mouse systemsthat are considered to be significantly different from the humancounterpart.

Abbreviations Used in the Present Application:

-   CTL, cytotoxic T lymphocyte-   VEGF, vascular endothelial growth factor-   P1GF, placenta growth factor-   VEGFR1, vascular endothelial growth factor receptor 1-   VEGFR2, vascular endothelial growth factor receptor 2-   TGM, transgenic mice-   TAA, tumor associate antigen.-   i.d., intradermal injection-   s.c., subcutaneous injection-   IFA, incomplete FREUND's adjuvant

BRIEF SUMMARY OF THE INVENTION

The present invention provides peptides that induce cytotoxic T cellsagainst endothelial cells endogenously expressing VEGFR1. The peptidesof the invention comprise an amino acid sequence of SEQ ID NO: 1, 2, 13or a sequence wherein 1, 2, or several amino acids are substituted oradded. In certain embodiments, the second amino acid from the N terminusis leucine or methionine. In some embodiments, the C-terminal amino acidis valine or leucine.

The present invention also provides peptides comprising the amino acidsequence of SEQ ID NO: 32, or a sequence wherein 1, 2, or several aminoacids are substituted or added. In certain embodiments, the second aminoacid from the N terminus is phenylalanine, tyrosine, methionine, ortryptophan. In some embodiments, the C-terminal amino acid isphenylalanine, leucine, isoleucine, tryptophan, or methionine.

The present invention further provides pharmaceutical compositions fortreating or preventing tumors, wherein the composition comprises thepeptides of the invention.

The present invention provides exosomes that present on their surface acomplex comprising the peptide of this invention and an HLA antigen. Insome embodiments, the HLA antigen is HLA-A24 (e.g., HLA A2402) orHLA-A02 (HLA-0201).

Methods of inducing antigen-presenting cells having high cytotoxic Tcell inducibility and methods of inducing cytoxic T cells comprisingadministering the peptides of the invention to a patient are alsoprovided. In some embodiments, the methods comprise transferring a genecomprising a polynucleotide encoding the peptide of the invention toantigen-presenting cells. The invention provided isolated cytotoxic Tcells and antigen presenting cells which are induced by the methods ofthe invention. The present invention provides antigen-presenting cells,which comprise a complex formed between an HLA antigen and the peptideof the invention.

The present invention also provides vaccines for inhibiting angiogenesisat a diseased site, wherein the vaccine comprises the peptide of theinvention as the active ingredient. The vaccine of the invention may beintended for administration to a subject whose HLA antigen is HLA-A24 orHLA-A02. In some embodiments, the vaccine is used to suppress the growthof and/or metastasis of malignant tumors.

The present invention further provides methods of treating or preventingtumors in a subject comprising administering to said subject a vaccinecomprising a peptide of the invention, or an immunologically activefragment, or a polynucleotide encoding the peptide.

The invention also provides methods of treating or preventingangiogenesis-mediated disease in a subject comprising administering avaccine of the invention. In some embodiments, the angiogenesis-mediateddisease is diabetic retinopathy, chronic rheumatoid arthritis,psoriasis, or atherosclerosis.

It is to be understood that both the foregoing summary of the inventionand the following detailed description are of a preferred embodiment,and not restrictive of the invention or other alternate embodiments ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the establishment of HLA-A*0201 restricted CTLclones using epitope candidates derived from VEGFR1. Cytotoxicity ofeach CTL clone against T2 cells pulsed each epitope peptide binding withHLA-A*0201. T2 cells were used for CTL responses in the presence orabsence of each peptide. CTL clones showed specific cytotoxicitiesagainst the target cells pulsed with corresponding peptides. E/T ratioindicates effector/target-cell.

FIG. 2 is a graph showing the establishment of HLA-A*2402 restricted CTLclones using epitope candidates derived from VEGFR1. A24-LCL cells wereused for CTL responses restricted to HLA-A*2402 in the presence orabsence of each peptides binding with HLA-A*2402. CTL clones showedspecific cytotoxicities against the target cells pulsed withcorresponding peptides. E/T ratio indicates effector/target-cell.

FIG. 3 is a graph showing the cytotoxicity against endogeneously VEGFR1expressing cells. HLA-A*2402 CTL clone was examined for the cytotoxicityagainst VEGFR1 expressing cells (AG1-G1-Flt-1) and control (AG1-G1) witha 4-hr⁵¹Cr-release assay. These CTL clones showed the cytotoxicitiesagainst AG1-G1-Flt-1, but not against AG1-G1. E/T ratio indicateseffector/target-cell.

FIG. 4 is a graph showing the results of in vivo CTL response associatedwith the vaccination using VEGFR1-epitope peptides by IFN-γ ELISPOTassay. IFA-conjugated peptides were injected i.d. into A2/Kb TGM on day0 and day 11. On day 21, splenocytes of the vaccinated mice were used asthe responder cells, and T2 cells pulsed with or without peptides wereused as the stimulator cells for ELISPOT assay. Specific production ofIFN-γ for the corresponding peptide was observed in the mice vaccinatedwith VEGFR1-1087, -770, -417 peptides. (E/T ratio: x20).

FIG. 5 is a graph showing the results of in vivo inhibition oftumor-induced angiogenesis. The angiogenic responses induced by MC38cells in A2/Kb TGM. The mice were vaccinated twice with HBSS, IFA alone,and VEGFR1-peptide conjugated with IFA (VEGFR1-1087, -770, -417).Differences were visible macroscopically in the implanted chambersremoved from s.c. fascia of vaccinated mice. Quantification of newlyformed vessels in the angiogenic response. Significant inhibition oftumor-induced angiogenesis was observed in mice vaccinated withVEGFR1-1087, -770, -417 peptides. Error bar indicate s.e.

FIG. 6 is a graph showing the results of in vivo anti-tumor effect.A2/Kb TGM was inoculated i.d. with MCA205 cells. HBSS, IFA only, and IFAconjugated with VEGFR1-1087, -770, -417 peptides were vaccinated 4 daysand 11 days later (the indicated arrow). Significant suppression oftumor growth was observed with the vaccination using VEGFR1-1087, -770peptides conjugated with IFA.

DETAILED DESCRIPTION OF THE INVENTION

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

Angiogenesis has been shown to be a critical mechanism for tumorprogression. Multiple studies have suggested that tumor growth can besuppressed if tumor angiogenesis can be inhibited using various types ofanti-angiogenic agents. In the present invention, we examined thepossibility of developing novel immunotherapy targeting VEGFR1. We firstidentified the peptide epitopes of VEGFR1 from HLA-A2 and A24, andsuccessfully established CTL clones with potent cytotoxicity againstendothelial cells endogenously expressing VEGFR1. In A2/Kb transgenicmice, vaccination using these epitope peptides in vivo was associatedwith significant suppression of tumor growth in a therapeutic model. Inanti-angiogenesis assay, tumor-induced angiogenesis was significantlysuppressed with the vaccination. These results in vitro and in vivostrongly suggest VEGFR1 could be a promising target, and support thedefinitive rationale of the clinical development for anti-angiogenicimmunotherapy against various kinds of cancers.

In the present invention, we examined the effectiveness of this novelimmunotherapy in systems closely related to clinical settings. Weidentified the epitope peptides of human VEGFR1 restricted to HLA-A*0201and A*2402 (Rammensee et al., 1995. Immunogenetics. 41: 178-228.) andshowed that CTLs induced with these peptides have potent and specificcytotoxicity against not only peptide-pulsed target cells but alsotarget cells endogenously expressing VEGFR1 in an HLA class I restrictedfashion. Furthermore, we examined in vivo anti-tumor effects of thevaccination with these epitope peptides using a unique mouse model thatmay be directly translated into the clinical setting. Our model systemuses A2/Kb transgenic mice (TGM), which have been shown to be useful forthe analysis of human CTL epitopes. There is approximately 71%concordance between humans and A2/Kb TGM in the CTL repertoire(Wentworth et al., (1996) Eur. J. Immunol. 26: 97-101.). To constructtumor systems, we transplanted syngeneic mouse tumor cells which werechemically induced in C57BL/6 mice (H-2 Kb) not expressing HLA-A*0201molecules. This tumor system, combining A2/Kb TGM and H-2 Kb mouse cellline, offers a unique setting. Since endothelial cells in A2/Kb TGMexpress HLA-A*0201 molecule, the CTLs induced by vaccination usingVEGFR1 epitope peptides recognize endothelial cells which express bothHLA-A*0201 and VEGFR1. Thus, in vivo anti-tumor effects of ananti-angiogenic vaccine can be evaluated in HLA-A*0201 restrictedfashion. However, they do not recognize tumor cells even if they expressVEGFR1 because they do not express HLA-A*0201. In this in vivo tumormodel, vaccination using these epitope peptides was associated withsignificant suppression of the tumor growth. In an anti-angiogenesisassay, tumor-induced angiogenesis was significantly suppressed withvaccination using these epitope peptides. These results show that thevaccination using epitope peptides derived from VEGFR1 induces anantitumor-immune response.

Identification of the tumor associate antigens (TAAs) has enabled theclinical development of peptide-based cancer vaccines, which couldinduce CTLs and lyse tumor cells in HLA class I restricted fashion(Bruggen et al., (1991) Science. 254: 1643-7, Boon et al., (1996) J.Exp. Med. 183: 725-9, Rosenberg et al., (1998) Nat. Med. 4: 321-7,Butterfield et al., (1999) Cancer Res. 59: 3134-42.). Multiple clinicaltrials using TAA peptides have reported that tumor regressions wereobserved in the melanoma patients (Rosenberg et al., (1998) Nat. Med. 4:321-7. Nestle et al., (1998) Nat. Med. 4: 328-32, Thurner et al., (1999)J. Exp. Med. 190: 1669-78, Belli et al., (2002) J. Clin. Oncol. 20:4169-80, Coulie et al., (2002) Immunol. Rev. 188: 33-42.). It has beensuggested that clinical efficacy could be effected by loss ordown-regulation of HLA class I molecules on the tumor cells (Cormier etal., (1998) Int. J. Cancer. 75: 517-24, Paschen et al., (2003) Int. J.Cancer. 103: 759-67, Fonteneau et al., (1997) J. Immunol. 159: 2831-9.Reynolds et al, (1998) J. Immunol. 161: 6970-6.). The frequency oftumors showing some alteration in expression of HLA class I moleculeshas been estimated to be more than 40% (Cormier et al., (1998) Int. J.Cancer. 75: 517-24, Paschen et al., (2003) Int. J. Cancer. 103:759-67.). Thus, significant portion of tumor cells could escape from theCTLs specific to the class I-epitope, even if CTLs could be successfullyinduced by cancer vaccine targeting tumor cells themselves. Thedevelopment of effective vaccines against endothelial cells involved intumor angiogenesis is an alternate approach. Endothelial cells aregenetically stable, do not show down-regulation of HLA Class Imolecules, and are critically involved in the progression of a varietyof tumor. Furthermore, the CTLs could directly cause damage to theendothelial cells without penetrating any other tissue, and lysis ofeven low numbers of endothelial cells within tumor vasculature mayresult in destruction of vessel integrity leading to inhibition of largenumbers of tumor cells (Folkman, J. (1995) Nat. Med. 1: 27-31.).Therefore, endothelial cells are a good target for cancer immunotherapy.To specifically and efficiently prevent tumor-angiogenesis with CTLresponse, the appropriate target needs to be selected among themolecules related to angiogenesis.

VEGF binds two related tyrosine kinase receptors, VEGFR1 (Flt-1)(SEQ IDNOS:44 and 45) and VEGFR2 (KDR), which are strongly expressed onendothelial cells in tumor tissue but not in normal tissue (Risau, W.(1997) Nature. 386: 671-4., Shibuya et al., (1999) Curr. Topics.Microbiol. Immunol. 237: 59-83., Plate et al., (1994) Int. J. Cancer.59: 520-9.). Suppression of these receptors showed anti-tumor effectsincluding neutralizing antibody, recombinant receptors or kinaseinhibitors (El-Mousawi et al., (2003) J. Biol. Chem. 278: 46681-91.,Stefanik et al., (2001) J. Neurooncol. 55: 91-100., Wood et al., (2000)Cancer Res. 60: 2178-89., Luttun et al., (2002) Nat. Med. 8: 831-40.,Lyden et al., (2001) Nat. Med. 7: 1194-201., Lu et al., (2001) CancerRes. 61: 7002-8.). Neutralizing anti-VEGFR1 antibodies efficientlyattenuated tumor growth and neovascularization with dose dependentmanner (Luttun et al., (2002) Nat. Med. 8: 831-40.). Furthermore,combination treatment with reagents blocking both VEGFR1 and VEGFR2 orthe use of a bifunctional antibody (‘diabody’) against VEGFR1 andVEGFR2, as such strategies resulted in stronger inhibition of vesselgrowth than monotherapy with a single antibody or with themonofunctional parent antibody (Lyden et al., (2001) Nat. Med. 7:1194-201., Lu et al., (2001) Cancer Res. 61: 7002-8.).

In the present invention using our novel model systems in vitro and invivo, we have demonstrated that an immunotherapy targeting tumor-inducedangiogenesis is effective. At first, we identified the epitope peptidesof VEGFR1 restricted to HLA-A*0201 and A*2402 which are frequentlyrecognized HLA-alleles (Rammensee et al., (1995) Immunogenetics. 41:178-228.). The CTLs were successfully induced with these peptides, andthey showed potent cytotoxic activities against not only peptide-pulsedtarget cells but also endogenously VEGFR1 expressing cells. Our findingsclearly demonstrated that human VEGFR1 is immunogenic in the humansystem.

Then, we demonstrated in vivo anti-tumor effects using multiple tumorcell lines and A2/Kb TGM, a good model system to evaluate immuneresponses in humans against tumor cells with the loss of HLA class Iexpression. It has been shown that there is approximately 71%concordance between the CTL repertoire of human and A2/Kb TGM (Wentworthet al., (1996) Eur. J. Immunol. 26: 97-101.). Thus, CTLs induced byvaccination using epitope peptides could recognize endothelial cells,which are derived from A2/Kb TGM and express VEGFR1 and HLA-A*0201, butdo not recognize the tumor cells which have no “human” MHC class Imolecules. Using this unique tumor model system, significant inhibitionof the tumor growth was observed with vaccination using these epitopepeptides. This peptide-based vaccine was also associated withsignificant suppression of tumors before the vaccination as well. Theseresults show that our vaccination strategy is effective even for tumorswith HLA deficit, which is considered to be one of the escape mechanismsof tumors. We have also shown in a DAS assay that tumor-inducedangiogenesis was significantly inhibited with vaccination using theseepitope peptides. This result shows that the inhibition of tumorangiogenesis can be achieved with peptide vaccination targeting themolecule expressed on tumor-induced vascular endothelial cells.

These results, in vitro and in vivo, show that VEGFR1 is a useful targetof immunological therapy using cellular immunity and support thedefinitive rationale of the clinical development of this strategyagainst a broad range of cancers.

Regarding HLA antigens, the data presented here show that the uses ofA-24 type or A-02 type (which are said to be highly expressed among theJapanese) are favorable for obtaining effective results. The uses ofsubtypes such as A-2402 and A-0201 are even more preferable. However, inthe clinic, the type of HLA antigen of the patient requiring treatmentis investigated in advance, which enables appropriate selection ofpeptides having high levels of binding affinity to this antigen, orhaving cytotoxic T cell (CTL) inducibility by antigen presentation.Furthermore, in order to obtain peptides showing high binding affinityand CTL inducibility, substitution or addition of 1, 2, or several aminoacids may be performed based on the amino acid sequence of the naturallyoccurring VEGFR1 partial peptide. Herein, the term “several” means 5 orless, or preferably 3 or less. Furthermore, in addition to peptides thatare naturally displayed, since the regularity of the sequences ofpeptides displayed by binding to HLA antigens is already known (Kubo RT, et al., (1994) J. Immunol., 152, 3913-24.; Rammensee H G, et al.,(1995) Immunogenetics. 41:178-228; Kondo A, et al., (1994) J. Immunol.155:4307-12.), modifications based on such regularity can be performedon the obtained peptides. For example, peptides showing high HLA-24binding affinity have their second amino acid from the N terminussubstituted with phenylalanine, tyrosine, methionine, or tryptophan, andpeptides whose amino acid at the C terminus is substituted withphenylalanine, leucine, isoleucine, tryptophan, or methionine may alsobe used favorably. On the other hand, peptides showing high HLA-0201binding affinity have their second amino acid from the N terminussubstituted with leucine or methionine, and peptides whose C-terminalamino acid is substituted with valine or leucine may be used favorably.Furthermore, 1 to 2 amino acids may be added to the N terminus and/or Cterminus of the peptide.

However, when the peptide sequence is identical to a portion of theamino acid sequence of an endogenous or exogenous protein having adifferent function, side effects such as autoimmune disorders orallergic symptoms against specific substances may be induced, therefore,preferably, situations in which the sequence matches the amino acidsequence of another protein is avoided by performing a homology searchusing available databases. Furthermore, if it is clear from homologysearches that not even peptides in which 1 or 2 amino acids aredifferent exist, there is no danger that modifications of theabove-mentioned amino acid sequence in order to increase the bindingaffinity with HLA antigens, and/or increase the CTL inducibility willcause such problems.

Although peptides having high binding affinity to the HLA antigens asdescribed above are expected to be highly effective as cancer vaccines,the candidate peptides, which are selected according to the presence ofhigh binding affinity as an indicator, must be examined for the actualpresence of CTL inducibility. Confirmation of CTL inducibility isaccomplished by inducing antigen-presenting cells carrying human MHCantigens (for example, B-lymphocytes, macrophages, and dendritic cells),or more specifically dendritic cells derived from human peripheral bloodmononuclear leukocytes, and after stimulation with the peptides, mixingwith CD8-positive cells, and then measuring the cytotoxic activityagainst the target cells. As the reaction system, transgenic animalsthat have been produced to express a human HLA antigen (for example,those described in BenMohamed et al., (2000) Hum. Immunol.; 61(8):764-79Related Articles, Books, Linkout.) may be used. For example, the targetcells can be radiolabeled with ⁵¹Cr and such, and cytotoxic activity canbe calculated from radioactivity released from the target cells.Alternatively, it can be examined by measuring IFN-γ produced andreleased by CTL in the presence of antigen-presenting cells that carryimmobilized peptides, and visualizing the inhibition zone on the mediausing anti-IFN-γ monoclonal antibodies.

As a result of examining the CTL inducibility of peptides as describedabove, those having high binding affinity to an HLA antigen did notnecessarily have high inducibility. Furthermore, nonapeptides ordecapeptides selected from peptides comprising the amino acid sequencesindicated by VLLWEIFSL (SEQ ID NO: 1), TLFWLLLTL (SEQ ID NO: 2),NLTATLIVNV (SEQ ID NO: 13), SYGVLLWEIF (SEQ ID NO: 32) showedparticularly high CTL inducibility.

Furthermore, the present invention provides immunogenic peptides of lessthan about 40 amino acids, often less than about 20 amino acids, usuallyless than about 15 amino acids having cytotoxic T cell inducibility, andcomprising the amino acid sequence of SEQ ID NO: 32 in which 1, 2, orseveral amino acids are substituted or added. In some preferredembodiments, the immunogenic peptide has an amino acid sequencecomprising 10 amino acids indicated in SEQ ID NO: 32 in which 1, 2, orseveral amino acids are substituted or added may have CTL inducibilityas long as it does not match the amino acid sequence of other proteins.In particular, amino acid substitution to phenylalanine, tyrosine,methionine, or tryptophan at the second amino acid from the N terminus,and to phenylalanine, leucine, isoleucine, tryptophan, or methionine atthe C-terminal amino acid, and amino acid addition of 1 to 2 amino acidsat the N terminus and/or C terminus are favorable examples. One of skillwill recognize that in addition to amino acid substitutions andadditions, immunologically active fragments of the peptides may also beused in the methods of the invention. Methods for determining activefragments are well known in the art.

The present invention also provides peptides having cytotoxic T cellinducibility, and comprising the amino acid sequence of SEQ ID NOS: 1,2, or 13, in which 1, 2, or several amino acids are substituted oradded. The amino acid sequence comprising 9 or 10 amino acids indicatedby SEQ ID NOS: 1, 2, or 13 in which 1, 2, or several amino acids aresubstituted or added may have CTL inducibility as long as it does notmatch the amino acid sequence of other proteins. In particular, aminoacid substitution to leucine, or methionine at the second amino acidfrom the N terminus, and to valine, or leucine at the C-terminal aminoacid, and amino acid addition of 1 to 2 amino acids at the N terminusand/or C terminus are favorable examples. One of skill will recognizethat in addition to amino acid substitutions and additions,immunologically active fragments of the peptides may also be used in themethods of the invention. Methods for determining active fragments arewell known in the art. CTL clones obtained by stimulation by thesemodified peptides can recognize the original peptides and cause damage.

Peptides of the invention can be prepared using well known techniques.For example, the peptides can be prepared synthetically, by recombinantDNA technology or chemical synthesis. Peptide of the invention may besynthesized individually or as longer polypeptides comprising two ormore peptides. The peptide are preferably isolated i.e., substantiallyfree of other naturally occurring host cell proteins and fragmentsthereof.

The peptides may contain modifications such as glycosylation, side chainoxidation, or phosphorylation; so long as the modifications do notdestroy the biological activity of the peptides as described herein.Other modifications include incorporation of D-amino acids or otheramino acid mimetics that can be used, for example, to increase serumhalf life of the peptides.

The peptides of this invention can be prepared into a combination, whichcomprises 1 or more peptides of the invention, for use as a cancervaccine that may induce CTL in vivo or ex vivo. The peptides may be in acocktail or may be conjugated to each other using standard techniques.For example, the peptides can be expressed as a single polypeptidesequence. The peptides in the combination may be the same or different.By administering the peptides of this invention, the peptides arepresented at a high density on the HLA antigens of antigen-presentingcells, then CTL that specifically react toward the complex formedbetween the displayed peptide and the HLA antigen are induced, andstrong immune response against vascular endothelial cells in the tumorcells is induced. Alternatively, antigen presenting cells that haveimmobilized the peptides of this invention on their cell surface areobtained by removing dendritic cells from the subjects, these arestimulated ex vivo by the peptides of this invention, CTL are induced inthe subjects by readministering these cells to the subjects, and as aresult, aggressiveness towards the target cells can be increased.

More specifically, the present invention provides immunogeniccompositions for treating tumors or preventing proliferation,metastasis, and the like of tumors. The compositions comprise 1 or morepeptides of this invention. The peptides may be the same or different inthe compositions. Furthermore, angiogenesis at the pathologic site isclosely linked to tumors, as well as diseases such as diabeticretinopathy, chronic rheumatoid arthritis, psoriasis, andatherosclerosis, and also to metastasis of solid tumors (Folkman, J,(1995) Nature Med. 1:27-31.; Bicknell et al., (1996) Curr. Opin. Oncol.8:60-5.). Therefore, the peptides of this invention can be used fortreating tumors, angiogenesis-mediated disease such as diabeticretinopathy, chronic rheumatoid arthritis, psoriasis, andatherosclerosis, as well as metastasis of solid tumors.

The peptides of this invention were found to inhibit the formation oftortuous blood vessels, which are morphologically different from normalblood vessels and are formed in malignant tumor tissues, and results ofanalyzing wound healing and fertility in vaccinated mice confirmed thatthere are no adverse effects on normal physiological angiogenesis.Furthermore, when cytotoxicity against non-proliferative orproliferative endothelial cells was tested in vitro using CTL clonesthat recognize the peptides of this invention, these clones were foundto show stronger activity towards proliferative endothelial cells thantowards non-proliferative endothelial cells. More specifically, theyfunction very specifically to disorders in which proliferativeendothelial cells are observed, and particularly to cancer.

In vivo and in vitro stimulation of dendritic cells by the peptides ofthis invention can be performed easily by exposing the cells to a highconcentration of the peptides so that these peptides exchange withpeptides that were originally immobilized on the cells. Therefore, thepeptides used in this invention must have at least a certain level ofbinding affinity to HLA antigens.

The peptides of this invention can be administered directly or as apharmaceutical composition that has been formulated by conventionalformulation methods. In such cases, in addition to the peptides of thisinvention, carriers, excipients, and such that are ordinarily used fordrugs can be included as appropriate without particular limitations. Theimmunogenic compositions of this invention may be used for treatment andprevention of various tumors such as gastric cancer, duodenal cancer,colon cancer, lung cancer, breast cancer, prostate cancer, and braintumor. The peptides of this invention targets the endothelial cells ofblood vessels that are newly formed in tumor tissues, and do not targetthe tumor cells themselves, therefore, a wide variety of tumors becometargets of treatment, and there are no particular limitations to theiruse.

Immunogenic compositions for treatment and/or prevention of tumors,which comprise the peptides of this invention as the active ingredients,can be administered with an adjuvant so that cellular immunity will beestablished effectively, or they may be administered with other activeingredients such as antitumor agents, and they may be administered byformulation into granules. An adjuvant that may be applied includesthose described in the literature (Johnson A G. (1994) Clin. Microbiol.Rev., 7:277-89.). Exemplary adjuvants include, aluminum phosphate,aluminum hydroxide, or alum. Furthermore, liposome formulations,granular formulations in which the drug is bound to few μm diameterbeads, and formulations in which a lipid is bound to the drug may beconveniently used. The method of administration may be oral,intradermal, subcutaneous, intravenous injection, or such, and systemicadministration or local administration to the vicinity of the targetedtumor is possible. The dose of the peptides of this invention can beadjusted appropriately according to the disease to be treated, age ofthe patient, weight, method of administration, and such, and isordinarily 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, morepreferably 0.1 mg to 10 mg, and is preferably administered once in a fewdays to few months. One skilled in the art can appropriately select thesuitable dose.

Alternatively, the present invention provides intracellular vesiclescalled exosomes, which present complexes formed between the peptides ofthis invention and HLA antigens on their surface. Exosomes can beprepared, for example by using the methods described in detail inPublished Japanese Translation of International Publication Nos. Hei11-510507 and 2000-512161, and is preferably prepared using antigenpresenting cells obtained from subjects who are targets of treatmentand/or prevention. The exosomes of this invention can be inoculated ascancer vaccines, similarly to the peptides of this invention.

The type of HLA antigens used must match that of the subject requiringtreatment and/or prevention. For example, for a Japanese, HLA-A24 orHLA-A02, particularly HLA-A2402 or HLA-0201 is often appropriate.

In some embodiments the vaccine compositions of the invention comprise acomponent which primes cytotoxic T lymphocytes. Lipids have beenidentified as agents capable of priming CTL in vivo against viralantigens. For example, palmitic acid residues can be attached to the ε-and α-amino groups of a lysine residue and then linked to an immunogenicpeptide of the invention. The lipidated peptide can then be administeredeither directly in a micelle or particle, incorporated into a liposome,or emulsified in an adjuvant. As another example of lipid priming of CTLresponses, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used toprime CTL when covalently attached to an appropriate peptide (see, e.g.,Deres, et al., (1989) Nature 342:561-4.).

The immunogenic compositions of the invention may also comprise nucleicacids encoding the immunogenic peptides disclosed here. See, e.g. Wolffet al., (1990) Science 247:1465-8; U.S. Pat. Nos. 5,580,859; 5,589,466;5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples ofDNA-based delivery technologies include “naked DNA”, facilitated(bupivicaine, polymers, peptide-mediated) delivery, cationic lipidcomplexes, and particle-mediated (“gene gun”) or pressure-mediateddelivery (see, e.g., U.S. Pat. No. 5,922,687).

The immunogenic peptides of the invention can also be expressed by viralor bacterial vectors. Examples of expression vectors include attenuatedviral hosts, such as vaccinia or fowlpox. This approach involves the useof vaccinia virus, e.g., as a vector to express nucleotide sequencesthat encode the peptide. Upon introduction into a host, the recombinantvaccinia virus expresses the immunogenic peptide, and thereby elicits animmune response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848. Anothervector is BCG (Bacille Calmette Guerin). BCG vectors are described inStover, et al., (1991) Nature 351:456-60. A wide variety of othervectors useful for therapeutic administration or immunization e.g.,adeno and adeno-associated virus vectors, retroviral vectors, Salmonellatyphi vectors, detoxified anthrax toxin vectors, and the like, will beapparent. See, e.g., Shata, et al., (2000) Mol. Med. Today 6:66-71;Shedlock, et al., (2000) J. Leukoc. Biol. 68:793-806; and Hipp, et al.,(2000) In vivo 14:571-85.

The present invention also provides methods of inducingantigen-presenting cells using the peptides of this invention. Theantigen-presenting cells can be induced by inducing dendritic cells fromthe peripheral blood monocytes and then contacting (stimulating) themwith the peptides of this invention in vitro or in vivo. When thepeptides of this invention are administered to the subjects,antigen-presenting cells that have the peptides of this inventionimmobilized to them are induced in the body of the subject.Alternatively, after immobilizing the peptides of this invention to theantigen-presenting cells, the cells can be administered to the subjectas a vaccine.

This invention also provides a method for inducing antigen-presentingcells having a high level of cytotoxic T cell inducibility, in which themethod comprises the step of transferring genes comprisingpolynucleotides that encode the peptides of this invention toantigen-presenting cells in vitro. The introduced genes may be in theform of DNAs or RNAs. For the method of introduction, without particularlimitations, various methods conventionally performed in this field,such as lipofection, electroporation, and calcium phosphate method maybe used. More specifically, it may be performed as described in Reeves ME, et al., (1996) Cancer Res., 56:5672-7; Butterfield L H, et al.,(1998) J. Immunol., 161:5607-13; Boczkowski D, et al., (1996) J. Exp.Med., 184:465-72; Published Japanese Translation of InternationalPublication No. 2000-509281. By transferring the gene intoantigen-presenting cells, the gene undergoes transcription, translation,and such in the cell, and then the obtained protein is processed andbinded to MHC Class I or Class II, and proceeds through a presentationpathway to present partial peptides.

Furthermore, the present invention provides methods for inducing CTLusing the peptides of this invention. When the peptides of thisinvention are administered to a subject, CTL is induced in the body ofthe subject, and the strength of the immune system targeting theangiogenic endothelial cells in the tumor tissues is enhanced.Alternatively, they may be used for an ex vivo therapeutic method, inwhich subject-derived antigen-presenting cells, and CD8-positive cells,or peripheral blood mononuclear leukocytes are contacted (stimulated)with the peptides of this invention in vitro, and after inducing CTL,the cells are returned to the subject.

Furthermore, the present invention provides isolated cytotoxic T cellsthat are induced using the peptides of this invention. The cytotoxic Tcells, which have been induced by stimulation from antigen-presentingcells that present the peptides of this invention, are preferablyderived from subjects who are targets of treatment and/or prevention,and can be administered by themselves or in combination with other drugsincluding the peptides of this invention or exosomes for the purpose ofantitumor effects. The obtained cytotoxic T cells act specificallyagainst target cells presenting the peptides of this invention, orpreferably the same peptides used for induction. The target cells may becells that express VEGFR1 endogenously, or cells that are transfectedwith a gene that encodes VEGFR1, and cells that present the peptides ofthis invention on the cell surface due to stimulation by these peptidescan also become targets of attack.

The present invention also provides antigen-presenting cells thatcomprise presentation of complexes formed between HLA antigens and thepeptides of this invention. The antigen-presenting cells that areobtained by contacting the peptides of this invention, or thenucleotides encoding the peptides of this invention are preferablyderived from subjects who are the targets of treatment and/orprevention, and can be administered as vaccines by themselves or incombination with other drugs including the peptides of this invention,exosomes, or cytotoxic T cells.

In the present invention, the phrase “vaccine” (also referred to as animmunogenic composition) refers to a substance that has the function toinduce immunity against tumor endothelial cells to suppress tumors uponinoculation into animals. According to the present invention,polypeptides comprising the amino acid sequence of SEQ ID NO: 1, 2, 13were suggested to be HLA-A02 restricted epitope peptides and SEQ ID NO:32 was suggested to be HLA-A24 restricted epitope peptides that mayinduce potent and specific immune response against tumor endothelialcells expressing VEGFR1. Thus, the present invention also encompassesmethod of inducing anti-tumor immunity using polypeptides comprising theamino acid sequence of SEQ ID NO: 1, 2, 13, 32. In general, anti-tumorimmunity includes immune responses such as follows:

-   -   induction of cytotoxic lymphocytes against endothelial cells in        tumors,    -   induction of antibodies that recognize endothelial cells in        tumors, and    -   induction of anti-tumor cytokine production.

Therefore, when a certain protein induces any one of these immuneresponses upon inoculation into an animal, the protein is decided tohave anti-tumor immunity inducing effect. The induction of theanti-tumor immunity by a protein can be detected by observing in vivo orin vitro the response of the immune system in the host against theprotein.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. A foreign substance that enters the livingbody is presented to T cells and B cells by the action of antigenpresenting cells (APCs). T cells that respond to the antigen presentedby APC in antigen specific manner differentiate into cytotoxic T cells(or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen,and then proliferate (this is referred to as activation of T cells).Therefore, CTL induction by a certain peptide can be evaluated bypresenting the peptide to a T cell by APC, and detecting the inductionof CTL. Furthermore, APCs have the effect of activating CD4+ T cells,CD8+ T cells, macrophages, eosinophils and NK cells. Since CD4+ T cellsare also important in anti-tumor immunity, the anti-tumor immunityinducing action of the peptide can be evaluated using the activationeffect of these cells as indicators.

A method for evaluating the inducing action of CTL using dendritic cells(DCs) as APC is well known in the art. DC is a representative APC havingthe strongest CTL inducing action among APCs. In this method, the testpolypeptide is initially contacted with DC and then this DC is contactedwith T cells. Detection of T cells having cytotoxic effects against thecells of interest after the contact with DC shows that the testpolypeptide has an activity of inducing the cytotoxic T cells. Activityof CTL against tumors can be detected, for example, using the lysis of⁵¹Cr-labeled tumor cells as the indicator. Alternatively, the method ofevaluating the degree of tumor cell damage using ³H-thymidine uptakeactivity or LDH (lactose dehydrogenase)-release as the indicator is alsowell known.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also beused as the APC. The induction of CTL is reported to be enhanced byculturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL hasbeen shown to be induced by culturing PBMC in the presence of keyholelimpet hemocyanin (KLH) and IL-7.

The test polypeptides confirmed to possess CTL inducing activity bythese methods are polypeptides having DC activation effect andsubsequent CTL inducing activity. Therefore, polypeptides that induceCTL against tumor endothelial cells are useful as vaccines againsttumors. Furthermore, APC that acquired the ability to induce CTL againsttumor endothelial cells by contacting with the polypeptides are usefulas vaccines against tumors. Furthermore, CTL that acquired cytotoxicitydue to presentation of the polypeptide antigens by APC can be also usedas vaccines against tumors. Such therapeutic methods for tumors usinganti-tumor immunity due to APC and CTL are referred to as cellularimmunotherapy.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction is known to increase by combining aplurality of polypeptides having different structures and contactingthem with DC. Therefore, when stimulating DC with protein fragments, itis advantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of anti-tumor immunity by a polypeptide canbe confirmed by observing the induction of antibody production againsttumors. For example, when antibodies against a polypeptide are inducedin a laboratory animal immunized with the polypeptide, and when growth,proliferation or metastasis of tumor cells is suppressed by thoseantibodies, the polypeptide can be determined to have an ability toinduce anti-tumor immunity.

The present invention also relates to a method of treating or preventingtumors in a subject comprising administering to said subject a vaccinecomprising a polypeptide encoded by a nucleic acid selected from thegroup consisting of VEGFR1 or an immunologically active fragment of saidpolypeptide, or a polynucleotide encoding the polypeptide or thefragment thereof. Administration of the polypeptide induces ananti-tumor immunity in a subject. Thus, the present invention furtherprovides a method for inducing anti tumor immunity. The polypeptide orthe immunologically active fragments thereof are useful as vaccinesagainst tumors. In some cases the proteins or fragments thereof may beadministered in a form bound to the T cell receptor (TCR) or presentedon an antigen presenting cell (APC), such as macrophage, dendritic cell(DC) or B-cells. Due to the strong antigen presenting ability of DC, theuse of DC is most preferable among the APCs.

Anti-tumor immunity is induced by administering the vaccine of thisinvention, and the induction of anti-tumor immunity enables treatmentand prevention of tumors. Therapy against or prevention of the onset oftumors includes any of the steps, such as inhibition of the growth oftumors cells, involution of tumors cells and suppression of occurrenceof tumors cells. Decrease in mortality of individuals having tumors,decrease of tumors markers in the blood, alleviation of detectablesymptoms accompanying tumors and such are also included in the therapyor prevention of tumors. Such therapeutic and preventive effects arepreferably statistically significant. For example, in observation, at asignificance level of 5% or less, wherein the therapeutic or preventiveeffect of a vaccine against tumors is compared to a control withoutvaccine administration. For example, Student's t-test, the Mann-WhitneyU-test or ANOVA may be used for statistical analyses.

The above-mentioned protein having immunological activity, or apolynucleotide or vector encoding the protein may be combined with anadjuvant. An adjuvant refers to a compound that enhances the immuneresponse against the protein when administered together (orsuccessively) with the protein having immunological activity. Examplesof adjuvants include cholera toxin, salmonella toxin, alum and such, butare not limited thereto. Furthermore, the vaccine of this invention maybe combined appropriately with a pharmaceutically acceptable carrier.Examples of such carriers are sterilized water, physiological saline,phosphate buffer, culture fluid and such. Furthermore, the vaccine maycontain as necessary, stabilizers, suspensions, preservatives,surfactants and such. The vaccine is administered systemically orlocally. Vaccine administration may be performed by singleadministration or boosted by multiple administrations.

When using APC or CTL as the vaccine of this invention, tumors can betreated or prevented, for example, by the ex vivo method. Morespecifically, PBMCs of the subject receiving treatment or prevention arecollected, the cells are contacted with the polypeptide ex vivo, andfollowing the induction of APC or CTL, the cells may be administered tothe subject. APC can be also induced by introducing a vector encodingthe polypeptide into PBMCs ex vivo. APC or CTL induced in vitro can becloned prior to administration. By cloning and growing cells having highactivity of damaging target cells, cellular immunotherapy can beperformed more effectively. Furthermore, APC and CTL isolated in thismanner may be used for cellular immunotherapy not only againstindividuals from whom the cells are derived, but also against similartypes of diseases in other individuals.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Any patents, patent applications, andpublications cited herein are incorporated by reference.

EXAMPLES

The present invention is illustrated in details by following Examples,but is not restricted to these Examples.

Materials and Methods

Cell Lines

The T2 cell line was generously provided by Dr. H. Shiku (Mie UniversitySchool of Medicine). The AG1-G1-Flt-1 and AG1-G1-Neo cell lines werekindly provided by Dr. M. Shibuya (Institute of Medical Science, TheUniversity of Tokyo). The AG1-G1 cell line was established from humanbenign hemangioma, and AG1-G1-Flt-1 was generated infecting the AG1-G0cell lines with the BCMGS plasmid vector carrying VEGFR1 cDNA. MCA205, amethylcholanthrene-induced murine fibrosarcoma cell line, was generousgifts from Dr. S. A. Rosenberg (National Cancer Institute, Bethesda,Md.). B16, a murine melanoma, and MC38, a murine colon adenocarcinomawere purchased from ATCC.

Synthetic Peptides

The candidates of VEGFR1 derived epitope peptides restricted toHLA-A*0201 (A2) and -A*2402 (A24) were selected based on the bindingaffinities to the corresponding HLAs. The binding affinities werepredicted with the website of BioInformatics & Molecular AnalysisSection (BIMAS) (Kuzushima et al., (2003) Blood. 101: 1460-8, Parker etal., (1994) J. Immunol. 152: 163-75.). These candidate peptides weresynthesized by Sawady Technology, Japan according to the standard solidphase synthesis method and purified by reversed phase HPLC. The purity(>95%) and the identity of the peptides were determined by analyticalHPLC and mass spectrometry analysis, respectively. The peptides used inthe present invention are listed in Table 1 and 2.

CEA peptide (DVLYGPDTPI (SEQ ID NO.41)) was used as a positive controlfor in vivo mouse model. CMV peptides (A02; NLVPMVATV (SEQ ID NO.42),A24; QYDPVAALF (SEQ ID NO.43)) were used as positive controls for humanCTL induction in vitro (Solache et al., (1999) J. Immunol. 163: 5512-8,Kuzushima et al., (2001) Blood. 98: 1872-81.).

Animals

The A2/Kb TGM, of which MHC class I consists of α1 and α2 domain ofHLA-A*0201 and α3 domain of mouse H-2 Kb, were prepared as describedelsewhere (Wentworth et al., (1996) Eur. J. Immunol. 26: 97-101.). Theanimals were maintained in the specific-pathogen-free Animal Facility ofthe Institute of Medical Science, The University of Tokyo, and all theprotocols for animal experiments were approved by the ethical committeeof our institute.

Generation of CTL Lines and Clones

Monocyte-derived dendritic cells (DCs) were used to induce CTL responsesagainst peptides presented on HLA as previously described (Nukaya etal., (1999) Int. J. Cancer. 80: 92-7, Tsai et al., (1997) J. Immunol.158: 1796-802, Nakahara et al., (2003) Cancer Res. 63: 4112-8.). Inbrief, the PBMCs were obtained from the healthy volunteers withcorresponding HLAs and cultured in the presence of GM-CSF (provided byKirin Brewery Company, Japan) and IL-4 (Genzyme/Techne, Minneapolis).After culture for 5 days, OK-432 (Chugai Pharmaceutical Corporation,Japan) was added to the culture to obtain mature DCs (Nakahara et al.,(2003) Cancer Res. 63: 4112-8.). On day 7, generated mature DCs werepulsed with each peptide for T cell stimulation. Using thesepeptide-pulsed DCs each time, the autologous CD8+ T cells werestimulated for three times on day 0, 7 and 14, and then the resultantlymphoid cells were tested for their cytotoxic activities on day 21.

To generate CTL clones, established CTL lines were plated in 96-wellplates at 0.3, 1, and 3 cells per well with allogenic PBMCs and A3-LCLas stimulator cells. Cytotoxic activities of resulting CTL clones weretested on the 14th day.

Cytotoxicity Assay

Cytotoxic activities were measured using a standard 4-h ⁵¹Cr-releaseassay. The T2 cells and A24-LCL were used as target cells pulsed withcandidate peptides. Percent specific lysis was calculated as follows:

${\%\mspace{14mu}{Specific}\mspace{14mu}{lysis}} = {\begin{bmatrix}{\begin{pmatrix}{{{experimental}\mspace{14mu}{release}} -} \\{{spontaneous}\mspace{14mu}{release}}\end{pmatrix}/} \\\begin{pmatrix}{{{maximum}\mspace{14mu}{release}} -} \\{{spontaneous}\mspace{14mu}{release}}\end{pmatrix}\end{bmatrix} \times 100.}$Immunogenicity of Epitope Peptides in A2/Kb TGM

For priming the peptide-specific CTLs, immunization was given using 200μl of vaccine mixture, which contains 100 μg of an HLA-A2 restrictedpeptide and 100 μl of IFA per mouse. The vaccine was injectedintradermally in the right flank for the first immunization on day 0 andin the other flank for the second on day 11. On day 21, splenocytes ofthe vaccinated mice were used as the responder cells, and T2 cellspulsed with or without peptides were used as the stimulator cells forELISPOT assay.

In Vivo Angiogenesis Assay

We examined the effects of peptide vaccination using dorsal air sac(DAS) assay which was designed to measure in vivo angiogenesis inducedby tumor cells as previously described (Oikawa et al., (1997) AnticancerRes. 17: 1881-6.). In brief, the A2/Kb TGM were vaccinated twice with1-week interval in the left flank using IFA conjugated correspondingpeptides as previously described with some modification (Schuler et al.,(1997) J. Exp. Med. 186: 1183-7, Song et al., (1997) J. Exp. Med. 186:1247-56, Specht et al., (1997) J. Exp. Med. 186: 1213-21.). Milliporechamber (Millipore Corporation, Bedford, Mass.) was filled with PBScontaining MC38 cells (1×10⁶ cells) and implanted in the dorsum ofanesthetized mice on day 0. The implanted chambers were removed froms.c. fascia on day 6 and then black rings were placed at the sitesexposed to a direct contact with the chamber. The angiogenic responsewas assessed with photographs taken using a dissecting microscope. Theextent of angiogenesis was determined with the number of newly formedblood vessels of >3 mm in length and scored semi quantitatively using anindex ranging from 0 (none) to 5 (many).

In Vivo Anti-Tumor Effects

We examined the anti-tumor effects of this vaccination with atherapeutic model. The 1×10⁵ MCA205 cells or the 5×10⁵ B16 cells wereinjected i.d. in the right flank on day 0, and vaccination was performedon day 4 and day 14 using IFA conjugated corresponding peptides.

Statistical Analysis

Each experiment was performed in triplicate to confirm reproducibilityof the results, and representative results are shown. Student's t testwas used to examine the significance of the data, when applicable. Thedifference was considered to be statistically significant when P valuewas less than 0.05.

Results

HLA Class I Binding Predicted Peptides from VEGFR1 Protein

The candidates of VEGFR1 derived epitope peptides restricted toHLA-A*0201 (A2) and -A*2402 (A24) were selected based on the bindingaffinities to the corresponding HLAs. The binding affinities werepredicted with the website of BioInformatics & Molecular AnalysisSection (BIMAS).

-   -   HLA Peptide Binding Prediction software:        (//bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform)        Kuzushima, K., et al., (2003) Blood. 101: 1460-8. Parker, K.C.,        et al., (1994) J. Immunol. 152: 163-75.        Establishment of CTL Clones Using Epitope Candidates Derived        from VEGFR1

We first tested the immunogenicity of VEGFR1 to determine the epitopepeptides. Epitope-candidate peptides were selected in the order of thebinding scores reflecting binding affinity of the peptide to the HLAclass I molecules (Table 1, Table 2).

TABLE 1 HLA-A*0201 binding predicted peptides from VEGFR1 protein StartSequence SEQ ID Binding Start Sequence SEQ ID Binding Position (9mer)No. affinity position (10mer) No. affinity 1087 VLLWEIFSL 1 1793 1153KLGDLLQANV 11 998 770 TLFWLLLTL 2 182 1029 LLSENNVVKI 12 167 1028ILLSENNVV 3 179 417 NLTATLIVNV 13 160 766 CVAATLFWL 4 137 1094SLGGSPYPGV 14 104 874 ALMTELKIL 5 75 1104 QMDEDFCSRL 15 96 861 KMLKEGATA6 47 1086 GVLLWEIFSL 16 92 875 LMTELKILT 7 38 797 IIMDPDEVPL 17 76 881ILTHIGHHL 8 36 1004 FQVARGMEFL 18 62 1027 NILLSENNV 9 35 220 YLTHRQTNTI19 48 220 YLTHRQTNT 10 34 590 ILLRTVNNRT 20 47

TABLE 2 HLA-A*2402 binding predicted peptides from VEGFR1 protein StartSequence SEQ ID Binding Start Sequence SEQ ID Binding position (9mer)No. affinity position (10mer) No. affinity 913 KYGNLSNYL 21 576 919NYLKSKRDLF 31 150 919 NYLKSKRDL 22 300 1084 SYGVLLWEIF 32 120 871EYKALMTEL 23 264 1001 SYSFQVARGM 33 35 1212 RYVNAFKFM 24 90 880KILTHIGHHL 34 17 1084 SYGVLLWEI 25 66 1003 SFQVARGMEF 35 17 1146RFAELVEKL 26 64 1212 RYVNAFKFMS 36 15 821 EFARERLKL 27 22 700 KIQQEPGIIL37 12 754 KSNLELITL 28 12 873 KALMTELKIL 38 12 819 KWEFARERL 29 12 1149ELVEKLGDLL 39 9 814 PYDASKWEF 30 11 1079 KSDVWSYGVL 40 8

We generated CTLs using these peptides and PBMCs given from healthyvolunteers with HLA-A*0201 and HLA-A*2402 as described in “Materials andMethods”, and CTL clones were successfully established.

These CTL clones showed specific cytotoxicity against the target cellspulsed with corresponding peptides (FIG. 1, FIG. 2).

We also examined the ability of established CTL clones induced withthese peptides to lyse the target cells endogenously expressing VEGFR1as well.

HLA-A*2402 CTL clone was examined for the cytotoxicity against VEGFR1expressing cells (AG1-G1-Flt-1) and control (AG1-G1) with a 4-hr⁵¹Cr-release assay. These CTL clones showed the cytotoxicities againstAG1-G1-Flt-1, but not against AG1-G1 (FIG. 3). The cytotoxicity wassignificantly blocked with mAbs against CD8 and HLA-class I, but was notblocked using mAbs against CD4 nor HLA-class II (data not shown).

In Vivo Anti-Angiogenesis and Anti-Tumor Effects Associated with theVaccination Using VEGFR1-Epitope Peptides.

We tested in vivo anti-angiogenesis effects and anti-tumor effects ofvaccination with VEGFR1-epitope peptides using A2/Kb TGM.

At first, we evaluated the immunogenicity of the epitope peptides forA2/Kb TGM to examine the specific production of IFN-γ of the CTLsinduced with these peptides by ELISPOT assay (FIG. 4). IFA-conjugatedpeptide was injected s.c. into A2/Kb TGM on day 0 and day 11. On day 21,splenocytes of the vaccinated mice were harvested and used as theresponder cells, and T2 cells pulsed with or without peptides were usedas the stimulator cells for ELISPOT assay. Specific production of IFN-γfor the corresponding peptide was observed in the mice vaccinated withVEGFR1-1087, -770, -417 peptides. In this ELISPOT assay using A2/Kb TGMsystem, positive results were shown for the epitope peptides identifiedusing human PBMCs.

We examined whether the vaccination using peptide derived from VEGFR1suppress the tumor-induced angiogenesis. To confirm the effects of thepeptide vaccination on angiogenesis induced by tumor cells, we employeddorsal air sac assay (DAS assay) which visualizes the extent ofneo-vascularization in vivo. In this semiquantitative assay, significantinhibition on angiogenesis was observed in the mice vaccinated withVEGFR1-1087, -770, -417 peptides (FIG. 5).

The vaccination using the epitope peptide showed strong antitumor effectin therapeutic model. The MCA205, a methylcholanthrene-induced murinefibrosarcoma cell line were injected i.d. into A2/Kb TGM on day 0, andvaccination was performed on these mice on 4 and 14 days after the tumorchallenge using VEGFR1-1087, -770, -417 peptides conjugated with IFA(FIG. 6). Significant suppression of tumor growth was observed with thevaccination using VEGFR1-1087, -770 peptides conjugated with IFA.Furthermore, significant inhibitions of tumor growth were observed invarious tumor cells (data not shown).

These results strongly suggest that the anti tumor effects induced withthe vaccination using the peptides derived from VEGFR1 might be mediatedby the inhibition of tumor-angiogenesis. Thus, vaccination with epitopepeptides derived from VEGFR1 could affect the growth of the tumor cellsthrough the effects on the VEGFR1-expressing endothelial cells of thevessels which support the tumor growth in vivo in this A2/Kb TGM-tumorsystem.

Discussion

Identification of the tumor associate antigens (TAAs) has enabled theclinical development of peptide-based cancer vaccine, which could induceCTLs and lyse tumor cells in HLA class I restricted fashion (Bruggen etal., (1991) Science. 254: 1643-7, Boon et al., (1996) J. Exp. Med. 183:725-9, Rosenberg et al., (1998) Nat. Med. 4: 321-7, Butterfield et al.,(1999) Cancer Res. 59: 3134-42.). Until now, multiple clinical trialsusing TAA peptides have reported that tumor regressions were observed inapproximately 20% rate of the melanoma patients. However, completeresponse has rarely been reported (Rosenberg et al., (1998) Nat. Med. 4:321-7. Nestle et al., (1998) Nat. Med. 4: 328-32, Thurner et al, (1999)J. Exp. Med. 190: 1669-78, Belli et al., (2002) Parmiani. J. Clin.Oncol. 20: 4169-80, Coulie et al., (2002) Immunol. Rev. 188: 33-42.).One of the possible reasons of modest clinical efficacy could be loss ordown-regulation of HLA class I molecules on the tumor cells (Cormier etal., (1998) Int. J. Cancer. 75: 517-24, Paschen et al., (2003) Int. J.Cancer. 103: 759-67, Fonteneau et al., (1997). J. Immunol. 159: 2831-9,Reynolds et al., (1998) J. Immunol. 161: 6970-6.). The frequency oftumors showing some alteration in expression of HLA class I moleculeshas been estimated to be more than 40% (Cormier et al., (1998) Int. J.Cancer. 75: 517-24, Paschen et al., (2003) Int. J. Cancer. 103:759-67.). Thus, significant portion of tumor cells could escape from theCTLs specific to the class I-epitope, even if CTLs could be successfullyinduced by cancer vaccine targeting tumor cells themselves. Theseproblems can be overcome with the development of effective vaccineagainst tumor angiogenesis, since endothelial cells are geneticallystable, do not show down-regulation of HLA Class I molecules, and arecritically involved in the progression of a variety of tumors.Furthermore, the CTLs could directly cause damage to the endothelialcells without penetrating any other tissue, and lysis of even lownumbers of endothelial cells within tumor vasculature will result indestruction of vessel integrity leading to inhibition of large numbersof tumor cells (Folkman, J. (1995) Nat. Med. 1: 27-31.). Therefore,endothelial cells are a good target for cancer immunotherapy. Tospecifically and efficiently prevent tumor-angiogenesis with CTLresponse, the appropriated target needs to be selected among themolecules related to angiogenesis.

The results presented here, in vitro and in vivo, demonstrate thatVEGFR1 can be used as target of immunological therapy using cellularimmunity and support the definitive rationale of the clinicaldevelopment of this strategy against a broad range of cancers.

INDUSTRIAL APPLICABILITY

The present invention provides novel peptides, which induce cytotoxic Tcells by targeting endothelial cells formed in a wide range of tumortissues, and are extremely effective as cancer vaccines. The presentinvention also provides immunogenic compositions comprising thesepeptides for treating and preventing tumors.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

1. An isolated peptide consisting of the amino acid sequence of SEQ IDNO:
 25. 2. A composition comprising the peptide of claim 1.